1. Thesis for doctoral degree (Ph.D.)
2010
The effect of therapeutic and
Nd:YAG laser as an adjunct
treatment modality in
periodontal therapy
Talat Qadri
Thesisfordoctoraldegree(Ph.D.)2010TalatQadriTheeffectoftherapeuticandNd:YAGlaserasanadjuncttreatmentmodalityinperiodontaltherapy
2. From THE DIVISION OF PERIODONTOLOGY,
DEPARTMENT OF DENTAL MEDICINE
Karolinska Institutet, Stockholm, Sweden
THE EFFECT OF
THERAPEUTIC AND ND:YAG
LASER AS AN ADJUNCT
TREATMENT MODALITY IN
PERIODONTAL THERAPY
Talat Qadri
Stockholm 2010
4. Dedication
To my mother.
She died on the day that I received my DDS degree. She struggled with her disease until
she knew that her son had finished his education and became a dentist.
5. Abstract
Laser irradiation has been proposed as an adjunct to conventional scaling and root planing
in the treatment of periodontitis. However, the reported outcomes of studies to date are
contradictory and the literature provides limited evidence to support an additional benefit
of laser application. The overall aim of the present thesis was to explore the potential of
adjunctive application of therapeutic and surgical lasers to improve treatment outcomes,
expressed in terms of clinical, radiographic and immunological parameters.
The present thesis is based on a series of four clinical studies of patients with moderately
severe periodontitis, treated by scaling and root planing. Two different types of dental
laser were investigated. Therapeutic lasers, which are claimed to stimulate cell
regeneration and boost the immune system, were investigated in studies I and II: the
general effect was investigated in Study I, while Study II compared the difference
between gas and diode lasers in the same spectrum, in order to evaluate the importance of
the length of coherence in biostimulation. In studies III and IV, the surgical Nd:YAG
laser, which is usually applied for sulcular debridement and pocket decontamination, was
evaluated in a novel approach. The test procedure comprised one single application of the
laser with water coolant after conventional scaling and root planing. In study III, the
outcome was evaluated after 3 months and in Study IV the long term outcome was
evaluated, at least one year post-treatment.
The split mouth design was used in all four studies. Study I showed a better clinical
outcome on the laser treated side and some improvement in immunological parameters.
The results of Study II support the hypothesis that a laser with a long length of coherence
is superior to one of a shorter length, although both lasers had some positive clinical
effect. In Study III a single application of the Nd:YAG laser as an adjunct to scaling and
root planing improved the short-term outcome and Study IV confirmed that this
improvement was sustained.
In conclusion, the results of these studies confirm the potential role of laser
irradiation as a non-invasive adjunctive to scaling and root planing in the treatment of
periodontitis.
Key words: Low level laser, Nd:YAG laser, protease activity, coherence length,
periodontal inflammation, cytokines, scaling and root planing.
6. LIST OF PUBLICATIONS
I. Qadri T, Miranda L, Tunér J, Gustafsson A. The short-term effects of low-
level lasers as adjunct therapy in the treatment of periodontal inflammation. J
Clin Periodontol. 2005;32:714-719.
II. Qadri T, Bohdanecka P, Tunér J, Miranda L, Altamash M, Gustafsson A.
The importance of coherence length in laser phototherapy of gingival
inflammation: a pilot study. Lasers Med Sci. 2007;22:245-251.
III. Qadri T, Poddani P, Javed F, Tunér J, Gustafsson A. A short-term evaluation
of Nd:YAG laser as an adjunct to scaling and root planing in treatment of
periodontal inflammation. J Periodontol. 2010;81:1161-1166.
IV.Qadri T, Javed F, Poddani P, Tunér J, Gustafsson A. Long-term effects of a
single application of a water-cooled pulsed Nd:YAG laser in supplement to
scaling and root planing in patients with periodontal inflammation. Lasers
Med Sci. 2010 Jun 27. [Epub ahead of print]
7. CONTENTS
Introduction 1
Laser light 1
Therapeutic lasers 3
Laser phototherapy (LPT) mechanisms 4
The Nd:YAG laser 5
The mechanisms underlying the Nd:YAG (surgical) laser 7
History of medical and dental applications of lasers 10
Safety and contraindications 12
Dosage 13
Laser phototherapy in Periodontology 14
Therapeutic lasers 16
Nd:YAG laser 18
Aims 19
General aims of the thesis 19
Specific Aims 19
Materials and Methods 20
Periodontal examination 20
Gingival crevicular fluid (GCF) 20
Laboratory Analyses 21
Radiographs 22
Statistical methods 22
The lasers used 24
Treatment methods 26
Results 28
Discussion 35
Overall Conclusions 43
Future perspectives 44
Acknowledgements 46
References 48
10. 1
INTRODUCTION
LASER LIGHT
The word LASER is an acronym for Light Amplification by Stimulated Emission of
Radiation. The first such device, a ruby laser, was introduced by Maiman in 1960
(http://laserstars.org/history/ruby.html). According to the European Standard IEC 601,
the definition of a laser is: Any device which can be made to produce or amplify
electromagnetic radiation in the wavelength range from 180 nm to 1 mm primarily by
the process of controlled stimulated emission . Laser light has two unique
characteristics: a very narrow band width and a high level of coherence.
Laser light is generally considered to be visible and collimated, i.e. travelling in a long,
straight line. This is true for many lasers: the most well-known collimated laser is the
laser pointer. However, medical lasers are generally neither collimated nor visible to
the naked eye. In surgery, as with the carbon dioxide laser (10600 nm), the beam can be
either focused for cutting or defocused for tissue ablation. Today lasers are widely
used, even in domestic appliances and are basic components of modern technology. In
medicine, lasers have been applied for decades in such diverse fields as surgery,
ophthalmology and blasting of kidney stones.
In physics, coherence is a property of waves that enables stationary (i.e. temporally and
spatially constant) interference. More generally, coherence describes all properties of
the correlation between the physical quantities of a wave. Two waves can combine to
create a larger wave (constructive interference) or detract from each other to create a
smaller wave (destructive interference), depending on their relative phase. Two waves
are said to be coherent if they have a constant relative phase (Figs.1,2).
(http://en.wikipedia.org/wiki/Coherence_%28physics%29).
The degree of coherence is measured by the interference visibility, a measure of how
perfectly the waves can cancel each other out by destructive interference. The beam
may or may not be parallel and the intensity can vary from a fraction of a milliwatt to
many watts. Coherence is reported to be important in biostimulation. It appears to have
11. 2
an additional positive effect in laser surgery, but the main advantage of surgical lasers
has little to do with the coherence.
Figure 1. Coherent light
Figure 2. Incoherent light
The length of coherence varies considerably between different types of lasers. The
shorter the bandwidth, the longer the length of coherence. The light from a gas-based
laser such as the HeNe (632.8 nm), has a coherence length directly from the tube of
many metres and a very narrow spectral bandwidth (Fig. 3). However, passage through
an optic fibre reduces the length of coherence considerably. Diode lasers, such as the
InGaAlP, can have a wavelength similar to the HeNe, but the length of coherence from
a laser diode is considerably shorter.
Figure 3. Spectral bandwidth of different light sources
From: Laser Therapy, Clinical Practice and Scientific Background. Prima Books AB,
2002
12. 3
THERAPEUTIC LASERS
The first commercialised biostimulative laser was a HeNe laser of less than 1 mW.
With its high degree of coherence the HeNe is an attractive laser for biostimulation but
limited by the need for an optic fibre, the size of the machine and the still rather low
power option, now typically in the range 5-25 mW. It has generally been replaced by
the InGaAlP laser, a diode producing red laser in the range 600 - 700 nm and able to
deliver as much as 500 mW. The most frequently used laser in dentistry is the GaAlAs
laser. It often operates in the spectrum between 780 and 830 nm. The 808 nm diode
dominates the market. Output is typically between 10 and 500 mW. An advantage of
the diode lasers is the small size and option for battery operation, making them rather
handy and portable. These lasers all work in continuous mode, but can be mechanically
or electronically pulsed ( chopped ). The optical penetration of the light varies with
several parameters. The short wavelengths in the red spectrum have less penetration
than those in the infrared spectrum. The type of tissue also influences the penetration.
Mucosa is rather transparent, bone and cartilage fairly transparent whereas penetration
into muscles is poor, due to the thickness of the tissue and the high vascularisation.
Blood is a major absorber of the light. Penetration also varies with distance from the
laser source to the target tissue: contact irradiation forces the light into the tissue, while
irradiation from a distance causes more reflection of the light.
The GaAs laser is different, being a superpulsed laser working at 904 nm. Superpulsed
lasers produce very powerful, pulsed peaks in the Watt range, but the duration of the
peak is typically only 200 nanoseconds. A GaAs laser presenting a Peak Power of 10
W typically has an average output of 10 mW. Pulsing is reported to be of importance in
biostimulation, but the evidence to date is based entirely on in vitro studies (Karu,
2007). Little is known of the role of pulsing in clinical application.
13. 4
Laser phototherapy (LPT) Mechanisms
To achieve an effect, the photon must be absorbed by photoreceptors. There are many
photoreceptors in the human body, e.g. the porphyrins. However, the most important
receptor has been identified as cytochrome c-oxidase, the terminal enzyme of the
Kreb s cycle. Cytochrome c-oxidase is an ATP producer (Passarella et al. 1984, Pastore
et al. 1996, Karu 2007). A cell in a reduced condition can be revitalized by stimulating
production of ATP. The laser light in the red spectrum severs the bond between NO
and cytochrome c-oxidase, allowing the enzyme to initiate production of ATP (Huang
et al. 2010). This production in itself leads to a cascade of events, such as increased
permeability of the cell wall and the Ca2
+ circulation. It has been speculated that
infrared laser light bypasses this process and acts directly on the cell membrane
permeability and the calcium ion channels. Cells in a normal redox situation are not
particularly responsive to LPT: the best effect is seen in cells in a reduced redox
situation (Almeida-Lopes et al. 2001). To date, studies of LPT have confirmed the
effects as natural processes and no effects outside the box have been reported.
14. 5
THE ND:YAG LASER
This type of laser produces light in a single crystal of Yttrium-Aluminium-Garnet with
the addition of - for example - elemental neodymium (Nd). The full name of this laser
is thus Neodymium-Yttrium-Aluminium-Garnet. Normally the laser is pumped by a
very strong flash lamp. A new type of Nd:YAG laser is the diode laser pumped
YAG:laser, in which instead of a flash lamp, powerful GaAlAs lasers are used to pump
optical energy to the Nd:YAG laser rod. The wavelength is 1064 nm. The light is
distributed via optical fibres, typically 300-600 micrometers in diameter.
The pulses are always in the millijoule (mJ) range and both the number of pulses per
second and the pulse length can be tailored by the operator to suit the intended target.
Most Nd:YAG lasers do not have a water cooling system.
The Nd:YAG lasers are in the watt (W) range. For dental use they are always pulsed,
each pulse providing a short energy in the millijoule range. The length of the pulse is
measured in nanoseconds (ns). Thus, the actual energy at the tips is determined by
several factors, such as basic output power, number of pulses per second and the pulse
length. These are often pre-programmed on the laser but can be chosen individually to
adapt to the situation or the experience of the operator. These parameters describe the
energy applied: the dose (energy density) is also influenced by the size of the optical
fibre. A thin fibre produces higher energy density at the tips: hence a 300 micron fibre
has an energy density four times greater than that of a 600 micron tip. The use of water
cooling will also influence the actual dose locally. Thus many parameters influence the
actual energy delivered. In this context, the technique adopted by the operator is also an
important determinant.
Modern dental Nd:YAG lasers are free-running and pulsed, in contrast to other
continuous wave lasers with gated pulse options. The ablative capacity is set either by
increasing the output power or the pulse repetition rate. The procedure is undertaken in
tissue contact mode and in constant motion.
For pulsed lasers, peak powers are orders of magnitude higher than average powers.
There are pronounced spikes, with peak power 1000 times higher than the average and
relatively long rest periods. Pulse width (the duration of each pulse) varies from 90 to
15. 6
1200 microseconds in different pulsed Nd:YAG lasers and is an important component
of this technology. The number of pulses (frequency, pulse repetition rate) per second
is one of the crucial variables in pulsed Nd:YAG lasers. With a high repetition rate
from 10 to 100 Hz in different devices, smoother cutting can be achieved at a very low
power setting, because the peak power in each pulse can be very high (White et al.
1994).
The 1064 nm wavelength is invisible, which complicates objective evaluation of the
actual effected area. Observation made by the author, using an infra-red camera
has revealed that the light is not concentrated around the fibre tip, but is spread like a
small sphere over a rather large area.
16. 7
The mechanisms underlying the Nd:YAG (surgical) laser
Nd:YAG laser energy is absorbed by tissue and it is this absorbance that allows surgical
excision and coagulation of tissue (Goldstein et al. 1995). Absorption by different
dental tissues is illustrated in Figure 5: absorption by hydroxyapatite is moderate. At
this wavelength, the ablative effect on hard dental tissue is obviously rather low. This
wavelength has a particular affinity for melanin or other dark pigments. Therefore dark-
pigmented microbes are more sensitive to this laser and can be eliminated at quite low
power settings, with no collateral damage to the adjacent tissue. The choice of
wavelength is important to reach a bactericidal effect. Harris & Yessik (2004)
developed a method for quantifying the efficacy of ablation of Porphyromonas
gingivalis (Pg) in vitro for two different lasers. The ablation thresholds for the two
lasers were compared in the following manner: Pg were cultured on blood agar plates
under standard anaerobic conditions. Haemoglobin is a primary absorber of the
wavelengths tested: thus in this context the blood agar simulated gingival tissue. Single
pulses of laser energy were delivered to the Pg colonies and the energy density was
increased until a small smoke plume was observed coincident with a laser pulse. The
energy density at this point was denoted as the ablation threshold. Ablation thresholds
to a single pulse were determined for Pg and for blood agar alone.
The investigation showed a major difference in ablation thresholds between the
pigmented pathogen and the host matrix for pulsed Nd:YAG, representing a significant
therapeutic window. Pg could be ablated without visible effect on the blood agar.
An 810 nm diode laser, on the other hand, destroyed both the pathogen and the gel.
Clinically, the pulsed Nd:YAG may selectively destroy pigmented pathogens, leaving
the surrounding tissue intact. The 810 nm diode laser may not demonstrate this
selectivity due to its longer pulse length and greater absorption by haemoglobin (Harris
& Yessik 2004).
It is postulated that the Nd:YAG laser eliminates primarily the dark-pigmented
microbes, such as Pg, whereas Aggregatibacter actinomycetemcomitans (Aa) which
17. 8
has no pigments, would not be similarly reduced. However, in a study by Andrade et al.
(2008) Aa was completely eliminated directly after irradiation, but had regained
approximately 50% of baseline level after 6 weeks. Such recurrence is reported in
several studies and is attributed to cross contamination from non-treated pockets and/or
saliva (Teughels et al. 2000).
The Nd:YAG laser has a certain biostimulative effect and this contributes to the
enhanced postoperative healing after Nd:YAG laser surgery. The energy densities in
the most peripheral zone (LPT) fall within the biostimulative range, as illustrated in
figure 4.
Carbonisation
Vaporisation
Laser tissue interaction
Coagulation
Laser beam
Denaturation
Photothermic effect
Photostimulating effect
LPT
Figure 4. Schematic illustration of the different light intensity zones (surgical lasers)
From: The New Laser Therapy Handbook, Prima Books AB, Grängesberg, 2010.
Courtesy: Edson Nagib
18. 9
Negative thermal effects of Nd:YAG laser have been reported from in vitro studies (Liu
et al. 1999, Israel et al. 1997). However, in vivo, effects on the root surface and the pulp
are not well-documented (Gaspirc 2001; Schwarz et al. 2008). The effect of laser
irradiation on the surrounding tissues is influenced by parameters such as power,
pulsing, fibre size, fibre angulations and cooling/no cooling. A study by White (1994)
suggested that powers between 0.3 3.0 W should not cause a damaging rise in
intrapulpal temperature. Likewise, Gold and Vilardi (1994) and Spencer (1996) also
reported that use of laser at 4 W is safe and does not damage the root surface.
Nd:YAG, which has little absorption in water, may be effectively cooled with
simultaneous air and water spray. Lasers with limited transmission through enamel and
dentine may also be effectively cooled by an air and water spray immediately after
lasing. Several studies have confirmed that application of an air and water spray
provides adequate heat protection to the pulp, comparable with cooling of the
conventional rotary bur (Miserendino et al. 1994). The absorption in different dental
tissues is illustrated graphically in figure 5.
Figure 5. The absorption spectrum for melanin, haemoglobin, enamel and water.
19. 10
HISTORY OF MEDICAL AND DENTAL APPLICATIONS OF LASERS
The first laser to be used in medicine was a ruby laser (wavelength 694 nm) and it was
soon applied in surgical procedures. The ruby is a solid state laser with a ruby rod as
the lasering medium. The first gas laser for surgery was the carbon dioxide (CO2) laser.
It had several appealing features in that it was able to remove superficial tissue without
harming the underlying tissues, due to the very high absorption of the 10600 nm in
water. Although this laser was expensive and large, it was soon accepted as a useful
tool in dental surgery, performing tissue ablation with a good degree of coagulation.
Conditions such as haemangiomas, leukoplakias and fibromas could easily be ablated
and malignancies could be removed surgically by focusing the beam. One of the first
Scandinavian papers on this topic was an animal study published by Luomanen (1987).
The Nd:YAG laser was also readily adopted in medicine, especially in the field of
ophthalmology. With a wavelength of 1064 nm, this laser could coagulate ocular
bleeding in diabetics, among other things. Myers (1991) was the first to apply the
Nd:YAG laser in dentistry: in fact, the first laser tested belonged to Myers brother, an
ophthalmologist. This laser proved useful for minor dental surgery, with a good
coagulatory effect. An unexpected observation was that little or no analgesia was
required. The laser could also be used to numb a tooth before drilling. Application as
a substitute for the dental drill attracted much public attention, but was not a great
success. To be absorbed into the dental hard tissues, a dark dye had to be applied to the
tooth before drilling and the process was very slow. It was not until the advent of the
Er:YAG lasers in the late 1990s that application of lasers for removal of hard dental
tissue became more widely adopted. These versatile lasers can penetrate dental hard
tissue at almost the same rate as a high-speed turbine drill. A major advantage is that
little or no analgesia is necessary. Laser-based methods have also been introduced as
aids for detection of early carious lesions, such as quantitative light-induced laser
fluorescence, using a diode laser with 655 nm (Tranaeus et al. 2005).
20. 11
The most recent additions to the dental laser family are the diode lasers. These typically
emit at wavelengths of 808, 940 or 980 nm, with outputs ranging from 3-7 watts. The
light is transmitted through an optical fibre. They are commercialised for soft tissue
management but are also used for endodontic decontamination and sulcular
debridement (Romanos et al. 2004). The diode lasers are much smaller than Nd:YAG
and Er:YAG lasers and less expensive.
Originally, the lasers introduced for medical application were all surgical in that they
were able to cut, evaporate and coagulate. However, another application was reported
very early by McGuff et al. (1965), studying the potential effect of the ruby laser on
tumours in hamsters. Different doses of ruby laser light were applied to various
tumours implanted in the animals cheek pouches. The results were unexpected: the
hamsters receiving laser light lived longer and even recovered completely, while none
of the control hamsters survived. The underlying mechanisms were not clarified and the
published papers do not appear to have attracted much attention. However, the results
were noted by the Hungarian surgeon Endre Mester (1967), who undertook some basic
experiments with a ruby laser on mice. The fur was shaved and wounds were created
bilaterally (Fig. 6). One side was irradiated with low doses of ruby laser and the other
side served as the control. Initially it was intended to increase the dose gradually, but it
was soon discovered that the irradiated wounds healed faster than the non-irradiated
wounds, while at higher doses the irradiation inhibited the wound healing. Even the
shaved fur grew back more quickly on the irradiated side. This was the first
documentation of the phenomenon of biostimulation . These lasers have then been
applied for a great variety of indications, such as radiation induced mucositis
(Bensadoun et al. 1999) and paresthesias of the inferior alveolar nerve (Khullar et al.
1996).
21. 12
Figure 6. Dorsal wounds on mice treated with ruby laser on the right side only
From: Laser Therapy, Clinical Practice and Scientific Background. Prima Books AB,
2002. Courtesy: Andrew Mester.
Safety and contraindications
The therapeutic lasers used in dentistry are classified as 3B, considered as low risk
devices and according to Swedish authorities (Strålskyddsmyndigheten - SSM) may be
used freely by anyone. Although the risk of eye injury is very low, protective goggles
are nevertheless recommended for the patient. There is no harmful heating of the tissue
when lasers are used in the recommended energy ranges. Since the limit of the ionising
radiation is around 320 nm, there is no risk of cancer induction in tissues.
None of several alleged contraindications have been verified during 40 years of use.
There are, however, some caveats. Due to the risk of stimulating malignant cells, laser
irradiation should not be used over known malignancies. However, the use of the
therapeutic laser is well documented for reducing the incidence of mucositis in patients
receiving chemo- and radiation therapy. Laser treatment is also contraindicated in
patients with coagulation disorders, because the effects of lasers on the mechanisms of
coagulation have yet to be determined.
22. 13
Dosage
To reach the dosage (also called fluence or energy density) the power of the laser must
be known. The power is expressed in milliwatts (mW). The energy delivered is a
function of the time. Thus, mW x seconds = energy. The energy is expressed in joules
(J). For instance, a laser of 100 mW used for 10 seconds delivers 1000 mJ = 1 J.
The dose is a function of the size of the irradiated area, expressed in cm2
. For instance,
if 1 J is applied to an area of 1 cm2
the calculation is 1 J/1 cm2
= 1 J/cm2
(dose).
However, if the irradiated area is 0.25 cm2
the calculation is 1 J/0.25 cm2
= 4 J/cm2
.
Another important factor in biostimulation is the power density, meaning the number of
mW over an area. If the laser emits 100 mW over an area of 1 cm2
, the calculation is
100/1 = 100 mW/cm2
. If the area is only 0.25 cm2
and receives the same number of
mW, the calculation is 100/0.25 = 400 mW/cm2
. In laser phototherapy, it is important
that all these variables are controlled, because each evokes different cellular reactions.
In the field of dentistry, the expression power density is quite familiar, because the
power of the dental curing light is expressed in mW/cm2
.
LPT follows the Arndt-Schultz law, (Fig. 7) which stipulates that for every substance,
small doses stimulate, moderate doses inhibit, and large doses destroy.
Figure 7. Arndt-Schultz law in phototherapy
From: Laser Therapy, Clinical Practice and Scientific Background. Prima Books AB,
2002
23. 14
LASER PHOTOTHERAPY IN PERIODONTOLOGY
Inflammation
Local inflammation is the central process in gingivitis and periodontitis. Acute
clinical manifestations include gingival swelling, redness and bleeding on probing.
Inflammation is basically a functional reaction necessary to protect the body from
bacterial invasion. Histologically an influx of leukocytes can be seen, primarily
neutrophils and monocytes/macrophages. When the inflammation becomes more
chronic the number of plasma cells and lymphocytes increases.
In the studies on which this thesis is based, clinical inflammation has been registered as
the Gingival Index (Silness & Löe 1964). This index assesses a combination of
swelling, redness and bleeding on probing. Changes in gingival pocket depth were also
measured: initially these reflect changes in the inflammatory condition. To complement
the clinical registration of inflammation, gingival crevicular fluid (GCF) volume has
been measured. GCF is an exudate/transudate that continuously flows out of the
gingival pocket. The volume increases with increasing inflammation and may thus be
considered a surrogate marker of inflammation, that is more objective than clinical
assessment of gingivitis (Golub & Kleinberg 1976).
To further assess the local inflammation a number of inflammatory mediators in GCF
have been analysed. Interleukin-1 (IL-1 ) is a proinflammatory cytokine that is
released by many different cells, among them macrophages. IL-1 can be considered a
general marker of the severity of inflammation in the tissues (Dinarello 2005). MMP-8
is a collagenase produced and released by several cells but mainly by neutrophilic
granulocytes during their migration from the blood capillaries to the inflamed tissues
(Sorsa et al. 2004). MMP-8 can thus be seen as an expression of neutrophil influx and
as such as a marker of inflammation. Elastase is a protease typical for
polymorphonuclear leukocytes (PMNL). It is mainly released from the neutrophils
during phagocytosis and may be regarded as an indicator of neutrophil activation
(Janoff 1985). IL-8 is a chemokine and an important inflammatory mediator released
from endothelial cells (Gamonal et al. 2000).
24. 15
In some cases the basically protective inflammatory response becomes tissue
destructive, i.e. periodontitis. The reasons for this change from a protective to a tissue
degrading inflammation is unclear but a Gram Negative anaerobic microflora together
with a susceptible host is probably necessary. The Swedish Council on Health
Technology Assessment estimates that signs of periodontitis are present in more than
40% of the Swedish adult population. Hugoson & Norderyd (2008) reported a 13%
incidence of severe periodontitis, although this is regional and age-related. Periodontitis
is more pronounced in those above the age of 40 years. Some forms of periodontitis are
very aggressive and may result in rapid loss of periodontal attachment and destruction
of alveolar bone. A major characteristic of the disease is the presence of bacteria in the
gingival pocket. Conventional therapy aims at reducing the bacterial load and
suppressing inflammatory signs through mechanical or chemical intervention,
sometimes including antibiotics. The outcome of mechanical treatment may be
compromised by the presence of furcations, invaginations and concavities. In these
cases there is a need for an additional treatment approach.
Periodontitis is primarily an inflammatory process which generally causes only minor
pain or discomfort. Thus scaling and root planing (SRP) are undertaken in order to
remove calculus and granulation tissue adhering to the root surface, and to create
conditions which facilitate maintenance of good oral hygiene. While SRP is considered
to be fundamental periodontal treatment, it is not always completely successful and
adjuvant therapies have been suggested.
In this context, laser therapy has been proposed, the goal being to target the
inflammation. However, to date the scientific basis for this treatment modality is not
well documented. The optimal parameters for each laser and for each particular
intervention have yet to be determined.
25. 16
Therapeutic lasers
Studies using therapeutic lasers have reported an effect on inflammation, mainly by
shortening the inflammatory process which in itself is essential for healing (Choi et
al. 2005, Pejcic et al. 2010). Sawasaki et al. (2009) and Silveira et al. (2008) reported
significantly increased mast cell degranulation after 670 nm laser irradiation of human
mucosa and gingiva, respectively. The degranulation leads to a release of histamine and
should theoretically stimulate an increased inflammatory response. It is speculated that
the increased mast cell degranulation accelerates the inflammatory process, which
eventually leads to wound healing via fibroblast proliferation and collagen synthesis.
Chronic periodontal inflammation leads to the destruction of the periodontal ligament
and subsequently to loss of alveolar bone. The latter is mediated primarily by
osteoclasts and triggered by the pro-inflammatory molecule Prostaglandin E2 (PGE2)
(Choi et al. 2005). There is some evidence in the literature that patients receiving LPT
in conjunction with conventional periodontal treatment experience improvement in
clinical inflammation (Pejcic & Zivkvic 2007).
Although gingivectomy is not a common procedure in modern periodontal therapy,
studies by Amorim et al. (2006) and Özcelik et al. (2008a) report improved healing
associated with application of 685 and 588 nm irradiation, respectively.
Garcia et al. (2009) compared LPT as an adjuvant to SRP for treatment of induced
periodontitis in rats. Treatment was compared to dexamethasone or saline solution.
Radiographic and histometric analysis showed less bone loss in animals treated with
SRP + LPT. A study by Pires de Oliveiro et al. (2008) has confirmed the stimulative
effect of LPT on osteoblasts. Özcelik (2008) has reported positive effects of LPT in
treating intra-bony defects with EMD enamel matrix protein derivate.
Periodontal wound healing is an important phase when the composition and integrity of
periodontal structures have been threatened by gingivitis, periodontitis or trauma. The
restoration of fibrous attachment and lost bone requires regeneration of destroyed
connective tissue, formation of new cementum and bone and attachment of new
26. 17
connective tissue fibres (Aukhil 1992). Thus successful repair involves several
processes, including inflammation and cellular migration, proliferation and
differentiation (Pitaru et al. 1994, Loevschall & Arnholt-Bindslev 1994).
Several in vitro studies have shown that LPT at certain wavelengths may stimulate
fibroblast proliferation, provided that certain combinations of exposure parameters and
power densities are used (Yu et al. 1994, Almeida-Lopes et al. 2001, Pereira et al.
2002, Azevedo et al. 2006). At higher energy densities, no effect or even decreased
proliferation has been reported (Kreisler et al. 2003). Therefore, Karu (1990) suggested
a window-specificity at certain wavelengths and energy densities, for which a
positive effect of laser phototherapy can be expected.
An important aspect of laser-tissue interaction is the coherence of the laser light. Many
studies have compared the biological effect of coherent and incoherent light and to
date all studies indicate a superior effect by lasers producing a long length of
coherence. Generally the comparisons have been made between lasers and Light
Emitting Diodes (LED). These light sources have a spectral width of 30-100 nm, while
the spectral widths of the lasers are in the range 0.01 1 nm. A study by Rosner et al.
(1993) investigated the effect of HeNe laser on regeneration of crushed optical nerves.
While HeNe laser delayed the degenerative process, non-coherent infrared light was
ineffective or affected the injured nerves adversely.
Coherence seems to be an important parameter in light stimulation of biological
scattering in bulk tissue. Karu et al. (1982, 1983) studied the importance of different
light characteristics in cell stimulation, such as wavelength, coherence, dose and time
regimens and concluded that coherence had no effect. However, in this context it is
important to note that these studies were conducted in vitro on monolayers of cells: the
cells were directly exposed to the laser and there was no scattering in the medium. As
the laboratory conditions do not simulate the clinical setting, the results should be
extrapolated with caution.
27. 18
Nd:YAG laser
Nd:YAG lasers have been used in periodontal treatment for many years but consensus
has yet to be reached about the general efficacy or the specific efficacy of different
power settings and clinical techniques. An important part of the laser device, which is
rarely discussed, is the optical fibre. Most bare fibres consist of a glass rod core made
of silica quartz with an outer surface cladding of different refractive index, and an outer
protective vinyl jacket. The standard options are diameters ranging from 200 to 600
micrometers. As the fibre diameter decreases, the energy densities increase and fibre
flexibility increases. Thin fibres are popular because of the high power density but less
than ideal for closed curettage, because they are prone to fracture and the energy
density is too high. The energy density of a 300 micrometer fibre is four times as high
as that of a 600 micrometer fibre. Thus, the use of a thin fibre in a closed area has
disadvantages. The high power densities will char areas in the pocket and carbonized
tissue will adhere to the tip. In the dark carbonized areas, absorption of the light
increases and so does heat. The aim of the laser treatment is not to use the instrument
for cautery, but to take advantage of the interaction between the light and the specific
tissue irradiated. Further to that, a thicker diameter makes the fibre stronger and
difficult-to-reach areas can be accessed more readily.
A major advantage of Nd:YAG laser periodontal therapy is that the procedure is
relatively pain free. From the patient s perspective this is certainly a major advantage.
The degree of pain is largely determined by the skill of the operator. However, in some
cases an analgesic gel or spray is advisable during the initial phase of the surgery. After
a while, it seems that the laser in itself provides an anaesthetic effect. Sulcular
debridement around hypersensitive teeth may sometimes be painful. In these cases, the
tooth crown can be irradiated from a short distance without water until an anesthetic
effect of the pulp is achieved. For the same reason, no water should be used when
hypersensitive tooth necks are treated with Nd:YAG laser. In combination with water
the area will be cleaned and the tubuli even more open. Without water there is the
potential for the laser to seal the tubuli (Lan & Liu 1996).
In general it can be stated that correctly applied, the lasers themselves are not
dangerous or damaging. It is the lack of knowledge that creates damage. The
undesirable side effects can vary primarily with power and energy density and secondly
with the type of laser used.
28. 19
AIMS
GENERAL AIMS OF THE THESIS
Several potential roles have been proposed for laser application in periodontal
treatment but the reported outcomes of studies to date are contradictory. The available
data are inadequate for recommendations with respect to optimal laser treatment
parameters.
The present thesis is based on a series of clinical studies of patients with moderately
severe periodontitis, treated by scaling and root planing. The studies were undertaken
with the overall aim of evaluating the potential of adjunctive application of therapeutic
and surgical lasers to improve the short and long-term treatment outcomes, expressed in
terms of clinical, radiographic and immunological parameters. Such studies are
essential in order to provide evidence on which to base recommendations for clinical
application.
Four studies were undertaken, the first two on therapeutic lasers and the third and
fourth studies on the Nd:YAG (surgical) laser.
SPECIFIC AIMS
The specific aims of the four studies were as follows:
Study I: to examine the effects of irradiation with laser phototherapy on inflamed
gingival tissue
Study II: to determine the possible influence of the length of coherence in laser
phototherapy
Study III: to compare the outcome of treatment of periodontitis by combined SRP and
a single application of water-cooled Nd:YAG laser irradiation with that of SRP alone
Study IV: a follow-up study of Study III, to determine whether the positive advantages
of the laser treatment were sustained over a longer time period
29. 20
MATERIAL AND METHODS
The following is a brief description of the materials and methods used in the four
studies. Detailed descriptions of the material and methods are presented in the original
papers (I-IV).
Periodontal Examination
Periodontal evaluation included PI (Plaque Index, Löe 1967) and GI (Gingival Index,
Silness & Löe 1964). PPD (Probing Pocket Depth) was measured with a graded
periodontal probe (PerioWise, Premier Dental, Plymouth Meeting, PA, USA ) at 4 sites
(mesial, distal, buccal and lingual). In studies I and II, the maxillary teeth, from 17 to
13 and 27 to 23 were registered. In studies III and IV, all the mandibular teeth, except
for the third molars, were registered.
Microbiological Examination
Subgingival plaque was harvested from the same site as GCF samples, by inserting
sterile paper points (size 30) for 30 seconds. The paper points from each side were then
pooled in sterile transport vials and sent to the laboratory for analysis. The subgingival
microbiota was analysed using a checkerboard DNA-DNA hybridization method
(Papapanu et al. 1997) and the frequencies of positive sites and of sites with cfu 10 6
were recorded. The following 12 micro-organisms were analysed: Porphyromonas
gingivalis, Prevotella intermedia, Prevotella nigrescens, Tannerella forsythensis,
Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Treponema
denticola, Peptostreptococus micros, Selenomonas noxia and Streptococcus
intermedia.
Gingival Crevicular Fluid (GCF)
In all subjects, two GCF samples were taken from each side of the maxilla, after
removal of supragingval plaque from the site to be sampled. The sites were isolated
with cotton rolls and gently dried with an air syringe before sampling. To collect GCF,
prefabricated paper strips (Periopaper, Oraflow Inc., Plainview, NY, USA) were
inserted until resistance was felt and removed after 30 seconds. GCF volume was
measured with a calibrated Periotron 8000 (Oraflow Inc). Samples were pooled and
30. 21
diluted in phosphate buffered saline (PBS) up to 1 ml. After elution for 15 minutes, the
strips were removed and the samples frozen at -20°C.
Laboratory analyses
Studies I and II
IL-1
The IL-1 content of the GCF samples was measured with sandwich ELISA, using a
monoclonal antibody (MAB 601, R&D Systems, Minneapolis, MN, USA) diluted 125
times in carbonate buffer, coated onto microtitre plates (Nunc Maxisorb Nanc A/S
Roskilde, Denmark) overnight at + 4 C. The plates were blocked with 1 % human
serum albumin (HAS) for 1 hour in room temperature. The detection antibody (BAF
201, R&D Systems), a biotinylated polyclonal goat antibody diluted 250 times, was
incubated for 45 min at 37°C. After washing, horseradish peroxidase conjugated
streptavidine, diluted 200 times in PBS +0.1% HSA, was added to the plates and
incubated in the same way as for the detection antibody.
The plates were washed again and the undiluted substrate (TMB, Sigma Chemical, St.
Louis, MO, USA) added. The reaction was stopped with 1M H2SO4 after 15 minutes.
Absorbency was read at 450 nm in a spectrophotometer (Millenia Kinetic Analyser,
Diagnostic Product Corporation, Los Angeles, CA, USA).
Elastase Activity
Total elastase activity was measured with a chromogenic substrate specific for
granulocyte elastase (Tanaka et al. 1990), (L-pyroglutamyl-L-propyl-L-valine-p-
nitroaniline, mw 445.5 Da, on a 96-well microtitre plate (Nunc Maxisorb, Nanc A/S).
After 2 h of incubation at 37 C, absorbency was read for a second time. The total
elastase activity is expressed in mAbs (milliabsorbances).
MMP-8 & IL-8
MMP-8 & IL-8 were analysed with commercial kits (Quantikine ®, R&D Systems
Inc.) in accordance with the manufacturer s instructions. A monoclonal antibody
specific for MMP-8 had been pre-coated on to a microplate. Samples diluted 10 times
31. 22
were pipetted into the wells and incubated at room temperature for 2 h. The plates were
then washed and a monoclonal antibody against MMP-8, conjugated to horseradish
peroxidase, was added and incubated again, as described previously. After another
washing procedure, the substrate solution was added and the reaction stopped after 15
min. with a stop solution. Within 20 min., the absorbency at 450 nm was read in a
spectrophotometer. The MMP-8 was expressed in ng and the amount of IL-8 in pg.
Study III
IL-1 , 4, 6, 8 and MMP-8
IL-1 , IL-4, IL-6 and IL-8 were analysed with Multiplex bead kits, using a Luminex
100 (Luminex Corp., Austin, TX, USA) and commercial immunoassays, Lincoplex
high-sensitivity human cytokine panel (Lincoplex/Millipore, St. Charles, MO, USA)
according to the manufacturer s instructions. The result was calculated with Bio-Plex
Manager software (Bio-Rad Laboratories, Hercules, CA, USA) and the cytokine levels
were determined as the total amount per site (pg) in the fluid. The collagenase MMP-8
was similarly analysed, but with a kit from R&D Systems (Abingdon, UK).
Radiographs
Digital bite-wing radiographs (Siemens, Bensheim, Germany) were taken with the
vertical long axis of the hemi-mandible using a software programme (Schick
Technologies Inc., NY, USA).
In Study IV all radiographs were taken by the author. Two observers recorded baseline
and post operative mandibular alveolar bone levels, in millimetres, at all approximal
surfaces, from the mesial of the second molar to the distal of the canine. Alveolar bone
loss was measured from the cemento-enamel junction (CEJ) to the most apical portion
of the alveolar bone. Teeth with suspected or obvious carious lesions at the CEJ were
not included.
Statistical methods
In studies I & II, statistical analyses were performed using Statistica 7 (Statsoft Inc. ,
2005, Tulsa, USA).
32. 23
In Study I, the significance of the differences in treatment effect between placebo and
laser was calculated with the Student paired t-test or the Wilcoxon signed rank test. The
frequencies of positive subjects and of subjects with 106
cfu of the analysed bacteria
were calculated with Fisher's exact test.
In Study II, the significance of the differences in treatment effect between the two
lasers was calculated with the Wilcoxon signed rank test.
In studies III and IV statistical analyses were performed using Statistica v.6.0
(Statsoft Inc. , 2005, Tulsa, USA).
In Study III, changes in the clinical parameters from baseline to follow-up, and between
the treatment modalities, were assessed for statistical significance using a paired t-test.
The laboratory data were analysed using the Wilcoxon signed rank test. Significance
was set at p<0.05.
In Study IV, the paired t test was applied to assess the changes in clinical parameters
from baseline to follow-up and between the treatment modalities. Normality was tested
with the Kolmogorov-Simirnov test.
33. 24
THE LASERS USED
Study I
A hand held, battery-operated Combi laser (Lasotronic AG, Baar, Switzerland) was
used. The device has two wave lengths that can be used together or separately. In this
study the wave lengths were utilized separately. Two lasers of identical appearance
were used in the study: (Fig. 8) one active and one placebo, the latter having only a
weak red LED diode instead of laser power. The active laser had two wavelengths, 635
and 808 nm, respectively. The output at 635 was 10 mW and at 808 nm 70 mW.
Figure 8. Active and placebo lasers
Study II
The lasers used in this study were a 3 mW HeNe laser 632.8 nm from Irradia AB,
Stockholm, Sweden and a Pocket Therapy diode laser, nominally 650 nm, from
Lasotronic AG, Baar, Switzerland (Fig. 9). Both had equal power of 3 mW.
Figure 9. The HeNe and the diode laser
34. 25
Studies III and IV
The laser used in Study III and IV was a Nd:YAG (Genius 9 SLD) laser, emitting
pulsed light 1064 nm, a fixed pulse repetition rate of 50 Hz , output from 1 W to 12 W
and coolant water and air levels available from 1 to 15. The fibre diameter was 600
micron (Genius Dental A/S, Tureby, Denmark).
Summary of the four studies
I Clinical study, double blinded
Split mouth
Clinical, immunological and bacteriological outcome
Plaque Index, Gingival Index,
Pocket Depth, Gingival
Crevicular Fluid, MMP-8, IL-
1ß, elastase, 12 bacterial
species
II Clinical study
Split mouth, double blinded
Clinical, immunological and bacteriological outcome
Plaque Index, Gingival Index,
Pocket Depth, Gingival
CrevicularFluid, MMP-8, IL-
8, elastase, 12 bacterial
species
III Clinical study, single blinded,
Split mouth
Clinical and immunological outcome
Plaque Index, Gingival Index,
Pocket Depth, Gingival
Crevicular Fluid, MMP-8, IL-
1ß, IL-4, IL-6, IL-8.
IV Clinical study, single blinded,
Split mouth
Radiological outcome
Plaque Index, Gingival Index,
Pocket Depth, Gingival
Crevicular Fluid, marginal
bone loss
35. 26
TREATMENT METHODS
Ethical Approval
These studies were approved by the regional ethical review board in Stockholm,
Sweden. All subjects gave their written informed consent before inclusion.
Study I
Seventeen patients with moderate periodontitis were included, 10 women and 7 men.
After clinical examination, all teeth were scaled and root planed (SRP). Oral hygiene
instructions were given and controlled at each session. Baseline measurements were:
Pocket Depth, Gingival Index and Plaque Index, all recorded before SRP. One week
after SRP, samples of gingival crevicular fluid (GCF) and subgingival plaque were
collected.
The laser therapy started one week later and continued once a week for 6 weeks. One
side of the upper jaw was treated with the active laser and the other with the placebo
unit.
The treated areas were:
(1) the buccal papillae, with 635 nm for 90 seconds (0.9 Joule, 4.5 J/cm2
, 50
mW/cm2
)
(2) 6 mm further apically, with 830 nm for 25 seconds (1. 75 Joules, 8.75 J/cm2
,
350 mW/cm2
)
(3) The sites were irradiated from both buccal and lingual aspects.
After the 6th week, the subjects underwent clinical re-examination, and new GCF and
plaque samples were collected.
Study II
The study sample comprised twenty patients with moderate periodontitis. After clinical
examination, all teeth were scaled and root planed (SRP). The dental hygienist now
started the laser therapy, once a week for 6 weeks. One side of the maxilla was treated
with HeNe laser and the other with a diode laser: choice of laser was determined by the
toss of a coin. Each dental papilla on the teeth 13, 14, 15, 16, 23, 24, 25 and 26 was
irradiated from the buccal aspect and 16 and 26 were also irradiated from the lingual
36. 27
aspect. All irradiated sites received 0.54 J of energy per session, total energy per
quadrant 3.25 J.
Studies III & IV
SRP + laser (SRPL) were used on one side of the mandible and the other was treated by
SRP alone. Thirty patients (13 males and 17 females) with a mean age of 50 years
(range 26 to 70 years) were originally included and randomly assigned to left or right
side. The treatment outcome was evaluated after 3 months.
The laser used in this study was a Genius 9 SLD Nd:YAG (Genius Dental A/S, Tureby,
Denmark), emitting pulsed light at a wavelength of 1064 nm. To avoid a thermal effect
while maintaining optimal therapeutic effect, the instrument was set at level-five,
giving the following parameters: average output 4 watt (W), energy per pulse 80
millijoule (mJ), pulse width 350 microseconds (µs), pulse repetition rate 50 Hertz (Hz),
pulse peak power 240 W, average power density at fibre end 1430 W/cm2
and peak
power density 85800 W/cm2
. Laser energy per treated tooth was 240 480 joules (J).
The fibre diameter was 600 µm (0.002826 cm2
). Water and air cooling were used
during irradiation. The time spent on each tooth varied between 60 to 120 seconds,
depending on accessibility.
The fibre was held in constant motion, in contact with the pocket epithelial lining
almost parallel to the long axis of the root. The power density and peak power density
reported above are calculated by a hypothetical 100% emission through the small fibre
tip. However, the energy is not emitted solely from the tip of the fibre; there is also
considerable lateral emission. Due to the high uncertainty about the total area of tissue
irradiated, the energy density (J/cm2
) was not calculated.
37. 28
RESULTS
None of the participants reported any adverse side effects that could be related to the
laser irradiation.
Study I
The results were as follows:
All clinical variables (PPD, PI, GI) showed greater reduction on the laser side (p<0.02).
The GCF volume decreased more on the laser side, -0.15 µl, compared to the placebo
side, -0.05 µl (p<0.02).
Figure 10. Box plot (above) shows the reduction in the clinical variables probing
pocket depth (PPD), plaque index (PI) and gingival index (GI) after SRP and an
additional treatment with laser or placebo. Filled boxes indicate the laser side.
38. 29
Table 1. Change in GCF volume (mean SD) and the laboratory variables (median
range) elastase activity, total amount of IL-1ß and MMP-8 in samples taken before and
after treatment with laser or placebo, n=17 patients
GCF Volume
µl
Elastase activity
mAbs
IL-1ß
pg
MMP-8
pg
Placebo -0.05 -9 (-576 - 252) -1.7 (57.9 - 24.7) 90 ((8180 - 5859)
Laser -0.15 32 (23 to 160) -0.8 (24.4 - 82.8) -70 (510 - 1145)
P-value 0.015* 0.15** 0.45** 0.052**
* p value calculated with the Student s paired t-test
** p-value calculated with Wilcoxon s signed rank test.
The concentration of MMP-8 increased on the placebo side and was somewhat reduced
on the laser side. The difference in treatment effect did not quite reach statistical
significance (p=0.052). No differences were disclosed between laser and placebo sides
with respect to elastase activity, IL-1 concentration or microbiological analyses.
39. 30
Study II
All clinical variables (PPD, PI, GI) showed greater reduction on the HeNe side (p-value
= 0.001).
P oc ket depth before and after las er treatm ent
P oc k et de pth before
Outlie rs
P oc k et de pth after
E xtrem e s
diod HeN e
Las er
-1
0
1
2
3
4
5
6
7
Figure 11. Box plot showing the reduction in the clinical variable probing pocket depth
after SRP and an additional treatment with HeNe or diode lasers. Filled boxes indicate
post treatment registrations.
Figure 12. Box plot showing the reduction in GCF volume after SRP and an additional
treatment with HeNe or diode lasers. Filled boxes indicate post treatment registrations.
40. 31
Figure 13. Box plot showing the clinical variables plaque index (PI), before and after
SRP and an additional treatment with HeNe or diode laser. Filled boxes indicate post-
treatment registrations.
Figure 14. Box plot showing the clinical variable gingival index (GI), before and after
SRP and an additional treatment with HeNe or diode laser. Filled boxes indicate post-
treatment registrations.
41. 32
Study III
Clinical outcomes
One week post-treatment, the PI (p<0.05), PPD (p<0.001) and GCF volumes (p<0.001)
on the irradiated side had decreased significantly compared to the control side. The GI
also decreased at the test side but the difference did not reach significance (Table 1).
The three-month follow-up confirmed that the improvements were sustained. The
treatment outcomes for the test side had improved significantly compared to the
control-site (PPD [p<0.01], GI [p<0.01], PI [p<0.01] and GCF volume [p<0.05]) (Table
2). During the three-month follow-up, the mean PPD decreased by 0.6 mm on the test
side compared to the control side.
43. 34
Study IV
Clinical and radiological results: At the follow up examination, PI (p<0.01), GI
(p<0.01) and PPD (p<0.001) were significantly lower on the test side than on the
control side. Radiological results showed a significant reduction in marginal bone loss
on the test side compared to the control side (p<0.05).
Gingival crevicular fluid volume: GCF volume was significantly lower on the test side
(mean change: -0.57 µl, range: -0.4 µl to 1.68 µl) than on the control side (mean
change: 0.15 µl, range: -0.12 µl to 1.11 µl) (p<0.01). These results are summarized in
Table 4 : clinical and laboratory outcomes.
44. -
35
DISCUSSION
Although lasers have been used in dentistry for many years, systematic reviews of the
literature report inadequate evidence to support their application in treatment of
periodontal disease. In the series of clinical studies on which this thesis is based, the
subjects comprised patients with moderately severe periodontitis, who underwent
conventional treatment by scaling and root planing. The split-mouth studies then
evaluated the potential of adjunctive application of therapeutic or surgical lasers to
improve the short and long-term treatment outcomes. Clinical, microbiological and
immunological parameters were recorded.
In the four studies undertaken, the first two using multiple applications of therapeutic
lasers and the third and fourth using a single application of the Nd:YAG (surgical)
laser, the overall results confirmed the beneficial effect of laser irradiation of the tissues
after scaling and root planing. Sites which received laser irradiation exhibited
improved clinical parameters and positive responses in terms of changes in
inflammatory markers in gingival crevicular fluid. Moreover, in Study IV, the long-
term outcome of a single application of the Nd:YAG laser also showed some gain in
alveolar bone levels.
The initial study in the series confirmed that as a complement to SRP, LPT can reduce
gingival inflammation. Adjunctive laser treatment resulted in significantly better
clinical variables such as PPD, PI, GI, and GCF than SRP alone. The decrease in
plaque index was also greater on the LPT side; this is in agreement with a study by
Iwase et al. (1989). Significant decreases in GI and PPD have also been reported by
Kiernicka et al. (2004). Ribeiro et al (2008) reported that LPT following SRP reduces
gingival inflammation and MMP-8 expression, while histological examination showed
a reduction in inflammatory cells. However, there are also some contradictory reports
on the effectiveness of LPT. Rydén et al (1994) and Yilmaz et al. (2002) reported that
LPT alone did not have an effect on the inflammatory response. Direct comparisons of
studies are however, difficult, due to differences in wavelengths, energy output and
irradiation mode. Further to that, Rydén et al. treated experimental gingivitis in healthy
individuals. Such experiments, using healthy animals or humans have recently been
45. 36
questioned. The genetically diabetic rat has, for instance, been a better model (al-
Watban et al.2007).
Study I showed that MMP-8 decreased on the LPT side and increased on the SRP-only
side, but the change did not reach significance (p=0.052). This in accordance with
studies by Luza & Hubacek (1996) and Fujimaki et al. (2003).
The microbiota were unchanged in both studies I and II. This may be due in part to the
timing of the sampling, after SRP, when the microbial load was already lowered. LPT
in itself does not have any bactericidal effect, but stimulation of macrophages (el Sayed
& Dyson 1996) could lead to phagocytosis and reduced bacterial load.
Ozawa et al. (1997) showed that LPT significantly inhibits the increase of plasminogen
activator (PA) induced in human periodontal ligament cells in response to mechanical
tension force. PA is capable of activating latent collagenase, the enzyme responsible for
cleaving collagen fibers. LPT was also efficient in the inhibition of PGE2 synthesis. In
human gingival fibroblast culture, LPT significantly inhibited PGE2 production
stimulated by lipopolysaccharide (LPS) through a reduction of COX2 gene expression
in a dose dependent manner. The decrease on PGE2 levels in cultures of primary human
periodontal ligament cells was also verified after cell mechanical stretching. Nomura et
al. (2001) verified that LPT significantly inhibited LPS-stimulated IL-1 production in
human gingival fibroblasts cells, and that this inhibitory effect was dependent on
irradiation time.
Safavi et al. (2008) evaluated the effect of LPT on gene expression of IL-1 , interferon
(IFN- ) and growth factors (PDGF, TGF- and bFGF) to provide an overview of
laser influence on their interactive role in the inflammatory process. The findings
suggest an inhibitory effect of LPT on IL-1 and IFN- production and a stimulatory
effect on PDGF and TGF- . These changes may be explained the anti-inflammatory
effects of laser and irradiation and its positive influence on wound healing.
Arany et al. (2007) in a study of the latent growth factor complex Transforming
Growth Factor-ß (TGF- ß), a multifaceted cytokine reported that the latent form can be
activated by LPT.
46. -
37
The findings of the above studies, describing different pathways of inflammatory
modulation, support the hypothesis explored in Study I that LPT can modulate the
periodontal inflammatory process, especially through the reduction of PGE2 release. In
summary, LPT influences the expression of COX2 and IL-1 , as well as MMP8,
PDGF, TGF- , bFGF and plasminogen. However, the capacity of LPT to modulate
inflammation does not seem to be confined to a single mechanism or to specific
wavelength, fluence or power: the different parameters tested in various studies gave
divergent results.
Study II demonstrated the importance of the coherence length of laser light. The
clinical signs of inflammation were significantly decreased on the HeNe laser side
(longer coherence length) compared to the diode laser side (short coherence length).
Several studies comparing the biological effects of coherent and non-coherent light
have reported that coherent light is superior (Hode 2005). In a study of regeneration of
crushed optical nerves, the HeNe laser delayed the degenerative process, while non-
coherent infrared light was ineffective (Rosner et al.1993). Similar conclusions have
been drawn from other studies (Haina et al. 1973, Rochkind et al. 1989). It is claimed
that coherent light is even more effective in deeper structures (Hode 2005). The cited
studies compared coherent and non-coherent light, which has in fact a coherence
length, albeit very minor. In Study II, two different coherent light sources of different
coherence length were compared. The results confirmed the hypothesis that coherence
length is an important determinant in laser phototherapy.
With respect to which wavelength best promotes cell proliferation, contradictory results
are reported. However, other factors besides wavelength and the energy dose are
important determinants of cell growth stimulation. Azevedo et al. (2006) tested two
power densities (428.57 and 142.85 mW/cm2
) at the same energy density (2 J/cm2
) and
showed that a lower power density caused higher stimulation. Moreover, the mode of
exposure, pulsing or continuous, may also play a role in optimizing stimulation. The
number of irradiation sessions and the treatment schedule will also influence the
outcome. The power densities used in studies I and II are low and impractical from a
clinical perspective. However, the design of the studies took into account
47. 38
recommendations in the literature (Huang et al. 2010), that the use of low power
densities over a longer treatment time would give an optimal outcome.
While pain is not a characteristic feature of chronic periodontitis, it is of major concern
after SRP. LPT application can decrease the pain sensation. However, the applied dose
must be considered closely. An approximate dose range of 2-6 J/cm2
is considered
optimal for wound healing and 6-10 J/cm2
for hastening the inflammatory process. A
shortening of the inflammatory process will in itself reduce the period of pain
perception. A larger dose will cause an inhibition of neural transmission and a rapid
decrease of pain (Chow et al. 2007). This dose is however, inhibitory for wound
healing and will prolong the inflammatory process. In this context, it is important that
the clinician understands the rationale underlying the laser application and is familiar
with appropriate dose ranges.
Disruption of collagen fibres in the periodontal ligament is attributed mainly to the two
collagenases MMP-1 and MMP-8. MMP-8 is released primarily from
polymorphonuclear leukocytes (PMNL) and secreted predominantly into the GCF: thus
MMP-8 levels in a GCF sample reflect the number of PMNL present and is an
expression of the severity of inflammation (Tervahartiala et al. 2000). IL-1 is a pro-
inflammatory cytokine released mainly from monocytes/macrophages, and is present in
the gingival tissues and GCF of patients with periodontal inflammation. Laser
irradiation is associated with significantly greater reductions in MMP-8 and IL-1 (Liu
et al. 1999).
Thus laboratory analyses confirm the clinical signs of improved healing at these sites.
The Liu studycited above compared the effects of SRP and SRP plus Nd:YAG laser on
the laboratory markers of periodontal inflammation. The six to 12 week follow-up
results showed a significant reduction in IL-1 levels after treatment with SRP plus
Nd:YAG laser compared to treatment by SRP alone. Similar results have been reported
by (Choi et al. 2004 and Ge et al. 2008).
The present studies disclosed no differences between SRP and SRP + laser irradiation
with respect to the cytokines IL- 1 and IL-8, 6, and 4, and the total amount of elastase
activity. Shimizu et al. (1995), in an in vitro study, reported that LPT affects the
48. -
39
production of cytokines. The discrepancy between in vitro and in vivo findings may be
attributable to the fact that in vitro the actual energy density at the target would be
considerably higher than in the clinical setting.
The relative effects of ultrasonic treatment, carbon-dioxide laser and Nd:YAG laser
have been investigated in several studies. Nd:YAG laser (without water-cooling) and
ultrasonic scaling resulted in significant improvements in clinical parameters (Israel et
al. 1997; Spencer et al. 1996; Miyazaki et al. 2006).
In contrast to the results of Study III, Sjöström and Friskopp (2002) using a similar
Nd:YAG laser, with water cooling, immediately following SRP, disclosed no
additional benefit for laser irradiation at the four-month control. A reduced need for
anaesthetics was the only obvious clinical advantage. The reason for the discrepant
results is unclear; however, it might be attributable to differences in the study design: in
the Sjöström study the laser was set to 7 W, in accordance with the manufacturer s
recommendations; whereas in Study III the setting was lower - 4 W.
A study by Lizarelli et al. (2006) showed that, within a limited range of power
Nd:YAG laser is a safe tool for irradiation of primary teeth in a broad range of
applications.
The laser fibre used in Study III was 600 µm in diameter and operated with a water
cooling system. Compared to a 600 µm tip, the power density of the conventional 300
µm tip is four times higher, causing greater carbonization and tissue adherence,
resulting in less control over the energy output at the tip. The 600 µm tip reduces the
power density and so does the water spray (Gold and Vilardi 1994; Radvar et al.1996).
In the present study, in order to overcome the loss of power at the fibre tip, the
following settings were selected: 4 W, 80 mJ per pulse, 50 Hz, and a pulse width of 350
µs. A further advantage of the 600 µm tip is the reduced risk of fibre fracture. Results
by Israel et al. (1997) showed that high energy, such as 9 W, can have negative effects
on the root surface. However, no such damage is associated with laser treatment at 4 W
and water coolant (Spencer 1996).
It is difficult to offer a comprehensive explanation for the greater improvement of
periodontal status at the laser-irradiated sites. An important contributory factor may be
49. 40
that laser application results in partial removal of the pocket epithelial lining. The
reduction in PI and PPD at the test sites might be associated with the improvement in
periodontal inflammation: because they experience less discomfort, patients may be
able to brush more thoroughly and maintain good oral hygiene at these sites.
The bactericidal effect of Nd:YAG laser has been tested in vitro by Kranendonk et al.
(2010). Suspensions of six different periodontal pathogens (Aggregatibacter
actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella
forsythia, Fusobacterium nucleatum and Parvimonas micra) were prepared in small
tubes and exposed to the Nd:YAG laser for five different intervals, using the following
laser settings: Power 6 Watt, Pulse Repetition Rate 50 Hz, Pulse duration 250 ms. After
exposure to the laser, aliquots of the suspensions were spread on blood agar plates for
bacterial counting. After 5 s of laser exposure, there was a decrease in total colony
forming units of all six selected micro-organisms. After laser irradiation for 15, 30 and
45 s, no viable bacterial cells remained.
In Study IV, sites irradiated with a single application of Nd:YAG laser as an adjunct to
SRP showed a reduction in periodontal inflammation and bone loss compared to the
control side. The improvement in clinical inflammation in terms of GI, was
corroborated by the reduction of GCF volume on the test compared to the control side.
Similar results have been reported previously (Wakao et al.1989) Laser irradiation has
been proposed as an adjunct to conventional scaling and root planning in the treatment
of periodontitis. However, the reported outcomes of studies to date are contradictory
and the literature provides limited evidence to support an additional benefit of laser
application. The overall aim of the present thesis was to explore the potential of
adjunctive application of therapeutic and surgical lasers to improve treatment
outcomes, expressed in terms of clinical, radiographic and immunological parameters.
The present thesis is based on a series of four clinical studies of patients with
moderately severe periodontitis, treated by scaling and root planing. Two different
types of dental laser were investigated. Therapeutic lasers, which are claimed to
stimulate cell regeneration and boost the immune system, were investigated in studies I
and II: the general effect was investigated in Study I, while Study II compared the
difference between gas and diode lasers in the same spectrum, in order to evaluate the
importance of the length of coherence in biostimulation. In studies III and IV, the
50. -
41
surgical Nd:YAG laser, which is usually applied for sulcular debridement and pocket
decontamination, was evaluated in a novel approach. The test procedure comprised one
single application of the laser with water coolant after conventional scaling and root
planing. In study III, the outcome was evaluated after 3 months and in Study IV the
long term outcome was evaluated, at least one year post-treatment.
The split mouth design was used in all four studies. Study I showed a better clinical
outcome on the laser treated side and some improvement in immunological parameters.
The results of Study II support the hypothesis that a laser with a long length of
coherence is superior to one of a shorter length, although both lasers had some positive
clinical effect. In Study III a single application of the Nd:YAG laser as an adjunct to
scaling and root planing improved the short-term outcome and Study IV confirmed that
this improvement was sustained.
Besides reducing periodontal inflammation laser irradiation has been proposed as an
adjunct to conventional scaling and root planning in the treatment of periodontitis.
However, the reported outcomes of studies to date are contradictory and the literature
provides limited evidence to support an additional benefit of laser application. The
overall aim of the present thesis was to explore the potential of adjunctive application
of therapeutic and surgical lasers to improve treatment outcomes, expressed in terms of
clinical, radiographic and immunological parameters.
Nd:YAG laser treatment also supports new connective tissue formation. A significant
reduction in PPD with increased clinical attachment levels is associated with Nd:YAG
laser therapy in patients with periodontitis (Yukna et al. 2007). This study
demonstrated new cementum and connective-tissue formation, also reported
subsequently by Romeo et al. (2009). Used at low energy, the Nd:YAG laser does not
cause damage to the cementum or the dental pulp. An earlier in vitro study by Radvar
et al. (1995) also showed that the Nd:YAG laser did not have a negative influence on
cementum, suggesting the formation of new connective tissues around the
periodontium.
New bone regeneration is a goal of periodontal therapy, but is seldom achieved. The
receptor activator of the nuclear factor-kB (RANK)/RANK ligand
(RANKL)/osteoprotegerin (OPG) system is essential in bone turn over. An animal
51. 42
study by Xu et al. (2009) investigated the effect of 650 nm irradiation on mRNA
expression of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin
(OPG) in rat calvarial cells. The authors concluded that the irradiation may directly
promote osteoblast proliferation and differentiation, and indirectly inhibit osteoclast
differentiation, by downregulating the RANKL:OPG mRNA ratio in osteoblasts. These
observations support an earlier study by Kim et al. (2007).
Study IV showed minor bone loss on the SRP only side while the side treated with laser
and SRP showed some bone gain. Similar results have been reported in a recent
experimental study in rats (de Almeida et al. 2008). While more bone regeneration is
reported in some clinical studies (Kim et al. 2010), in most such studies the selected
subjects exhibited more severe periodontitis at baseline, with pockets >4 mm, whereas
in the present series of studies the inclusion criteria stipulated that pocket depth should
not exceed 4 mm. Another difference in study design concerned the number of laser
applications: better bone regeneration was recorded in studies in which the subjects
underwent several laser therapy sessions, while the present studies III and IV included
only one session of Nd:YAG irradiation. While one such session may therefore not be
optimal, it appears to have been effective.
There are obvious weaknesses in Study IV, such as the small number of participants,
the relatively long unsupervised period and varying observation times, and the outcome
of only minor differences in alveolar bone height between the groups. A difference in
bone level of 0.18 mm is not clinically relevant. However, it is statistically significant
and shows that one application of Nd:YAG laser can have a long-term beneficial effect
on alveolar levels.
In conclusion, the results of these studies confirm the potential role of laser
irradiation as a non-invasive adjunctive to scaling and root planing in the treatment of
periodontitis.
Key words: Low level laser, Nd:YAG laser, protease activity, coherence length,
periodontal inflammation, cytokines, scaling and root planing.
52. -
43
OVERALL CONCLUSIONS
Study I showed that compared to SRP alone, additional treatment with LPT
significantly reduced periodontal gingival inflammation.
Study II showed that in laser phototherapy, a gas laser was more effective than a
diode laser in reducing gingival inflammation.
Study III showed that compared to SRP alone, an additional single application of
a water cooled Nd:YAG laser significantly improved clinical signs associated with
periodontal inflammation.
Study IV showed a long-term positive effect of a single application of Nd:YAG
laser in combination with SRP.
53. 44
FUTURE PERSPECTIVES
A review of the literature confirms that the outcome of laser applications in dentistry is
heavily dependent on the parameters selected. With sufficient knowledge, lasers can be
used for multiple applications and could be a substantial addition to the armamentarium
of the periodontist as well as the general dentist. But considering the great variability of
the available parameters, more research is necessary to identify therapeutic windows
for each indication and for each wavelength. Only then will dental lasers be more
readily accepted and sold in greater numbers, at prices that most dentists will consider
affordable. Researchers involved in this field have an obligation to be active in
education activities to ensure that dental lasers are applied in an evidence-based,
professional way. Future studies should preferably be multi-centre studies, where all
centres have identical equipment and methods. The present literature is difficult to
interpret due to lack of uniformity in selected parameters.
The reduction of the pocket microflora is an interesting topic. It is obvious that
Nd:YAG laser can reduce the bacterial burden, but to date there are few published
studies in this field.
In contrast to SRP, Nd:YAG laser can remove the pocket epithelial lining. The practical
importance of this property needs further verification. A negative outcome is not
necessarily attributable to lack of effect of the laser, but may be due to unsuitable
power settings, pulse repetition rates, total energy, treatment technique and fibre size.
The present series of studies highlights the importance of the fibre size. Further studies
are warranted to elucidate the influence of different fibre sizes on the clinical outcome.
The two Nd:YAG studies in this thesis have deliberately used a closed pocket mode, in
order to be able to compare the additional effect of the Nd:YAG laser after SRP.
However, a more surgical approach is also possible, where the pocket is opened during
the removal of the pocket epithelial lining, offering the operator a better view of the
pocket, allowing improved inspection of remaining debris. This technique also needs to
be investigated in future studies.
54. -
45
As therapeutic lasers and the Nd:YAG laser were investigated in this thesis and both
exhibited beneficial effects, a combined study would be of interest. After reducing the
bacterial load and the epithelial lining, a number of subsequent applications of LPT
could further improve healing by stimulating periodontal cells such as precursors to
osteoblasts. The adjuvant effect of LPT in traditional periodontal treatment modalities
such as GTR and organic and/or inorganic bone substitutes should also be highlighted.
The anti-inflammatory effect of LPT also needs to be better understood.
There are other lasers on the market such as diodes and Er:YAG. The application of
these in periodontology also warrants investigation.
Although the use of different lasers in periodontology has not been extensively
investigated, the literature suggests many potential advantages. Future research should
focus on establishing such an evidence-based treatment modality.
55. 46
ACKNOWLEDGEMENTS
Without the support and encouragement of many people it would not have been
possible to complete this thesis. Special thanks are due o all members of The
Department of Periodontology.
I am very grateful to my supervisor, Professor Anders Gustafsson, whose enthusiastic
guidance, support and encouragement enabled me to develop an understanding of the
subjects. Thank you Anders, for teaching me to manage and conduct scientific research,
and to write scientific papers.
I am grateful to Docent Lars Frithiof, Professor Björn Klinge and Dr Muhammad
Altamash for including me in the student exchange project .
I also wish to express my gratitude to Dr. Lars Hode, who has generously contributed
with assistance in the subjects bordering the physics.
I would like to thank Drs. Margareta Hultin, Tülay Lindberg and Kåre Buhlin for
encouragement and motivation.
Special thanks are also due to Kerstin Smedberg, former secretary and Heli Vänskä
secretary, Department of Periodontology, for kind support and help with administrative
matters.
I am grateful to Tommy Fredriksson and Marco Goytia Vásquez for their assistance
in computer software management.
I would like to thank Eva Hagström for participation in laser treatment of the study
subjects.
I would like to express my appreciation of the friendship and unfailing support of
Nikolas Christidis, Ai Komyyama, Lena Karlsson, Nilminie Rathnayake, Anna-
Kari Hajati, Fernanda Brito, Abier Sofrata and Sara Larsson.
56. -
47
Special gratitude is due to Dr. Jan Tunér and Gunilla Tunér for their kindness and
encouragement. Thank you for your continuous inspiration and encouragement.
Many thanks also to Niclas Lundin for sharing his photographic skills with me.
And last, but certainly not least, to my wife Anja and my daughters Kanwal and
Michiko for their great patience and generous support during the period of my PhD
studies.
57. 48
REFERENCES
Aimbire F, Albertini R, Pacheco M T, Castro-Faria-Neto H C, Leonardo P S,
Iversen V V, Lopes-Martins R A, Bjordal J M. Low-level laser therapy induces
dose-dependent reduction of TNFalpha levels in acute inflammation. Photomed Laser
Surg. 2006;24:33-37.
Almeida-Lopes L, Rigau J, Zangaro R A, Guidugli-Neto J, Jaeger M M.
Comparison of the low level laser therapy effects on cultured human gingival
fibroblasts proliferation using different irradiance and same fluence. Lasers Surg
Med. 2001;29:179-184.
Al-Watban F A, Zhang X Y, Andres B L. Low-level laser therapy enhances wound
healing in diabetic rats: a comparison of different lasers. Photomed Laser Surg.
2007;25:72-77.
Amorim J C, de Sousa G R, de Barros Silveira L, Prates R A, Pinotti M, Ribeiro
M S. Clinical study of the gingival healing after gingivectomy and low-level laser
therapy. Photomed Laser Surg. 2006;24:588-594.
Andrade A K, Feist I S, Pannuti C M, Cai S, Zezell D M, De Micheli G. Nd:YAG
laser clinical assisted in class II furcation treatment. Lasers Med Sci.
2008;23:341-347.
Arany P R, Nayak R S, Hallikerimath S, Limaye A M, Kale A D, Kondaiah P.
Activation of latent TGF-beta1 by low-power laser in vitro correlates with increased
TGF-beta1 levels in laser-enhanced oral wound healing. Wound Repair Regen.
2007;15:866-874.
Aukhil I. The potential contributions of cell and molecular biology to periodontal
tissue regeneration. Curr Opin Dent. 1992;2:91-96.
Azevedo L H, de Paula Eduardo F, Moreira M S, de Paula Eduardo C, Marques
M M. Influence of different power densities of LILT on cultured human fibroblast
growth: a pilot study. Lasers Med Sci. 2006;21:86-89.
58. -
49
Bensadoun R J, Franquin J C, Ciais G, Darcourt V, Schubert M M, Viot M.
Low-energy He/Ne laser in the prevention of radiation-induced mucositis. A
multicenter phase III randomized study in patients with head and neck cancer.
Supportive Care in Cancer. 1999;7:244-252.
Choi B K, Moon S Y, Cha J H, Kim K W, Yoo Y J. Prostaglandin E2 is a main
mediator in receptor activator of nuclear factor-kappaB ligand-dependent
osteoclastogenesis induced by Porphyromonas gingivalis, Treponema denticola, and
Treponema socranskii. J Periodontol. 2005;76:813-820.
Choi K H, Su I M, Kim C S, Choi S H, Kim C K. Effect of the carbon dioxide laser
on the clinical parameters and crevicular IL-1beta when used as an adjunct to gingival
surgery. J Int Acad Periodontol. 2004;6:29-36.
Chow R T, David M A, Armati P J. 830 nm laser irradiation induces varicosity
formation, reduces mitochondrial membrane potential and blocks fast axonal flow in
small and medium diameter rat dorsal root ganglion neurons: implications for the
analgesic effects of 830 nm laser. J Peripher Nerv Syst. 2007;12:28-39.
de Almeida J M. Theodoro L H, Bosco A F, Nagata M J, Oshiiwa M. G. In vivo
effect of photodynamic therapy on periodontal bone loss in dental furcations. J
Periodontol. 2008;79:1081-1088.
Dinarello C A. Interleukin-1beta. Crit Care Med. 2005; 33(12 Suppl): S460-462.
el Sayed S O, Dyson M. Effect of laser pulse repetition rate and pulse duration on
mast cell number and degranulation. Lasers Surg Med. 1996;19:433-437.
Fujimaki Y, Shimoyama T, Liu Q, Umeda T, Nakaji S, Sugawara K. Low-level
laser irradiation attenuates production of reactive oxygen species by human neutrophils.
J Clin Lasers Med Surg. 2003;21:165-170.
Gamonal J, Acevedo A, Bascones A, Jorge O, Silva A. Levels of interleukin-1 beta, -
8, and -10 and RANTES in gingival crevicular fluid and cell populations in adult
periodontitis patients and the effect of periodontal treatment. J Periodontol.
2000;71:1535-1545.
59. 50
Garcia V G, Fernandes L A, de Almeida J M, Bosco A F, Nagata M J, Martins T
M, Okamoto T, Theodoro L H. Comparison between laser therapy and non-surgical
therapy for periodontitis in rats treated with dexamethasone. Lasers Med Sci.
2010;25:197-206.
Gaspirc B, Skaleric U. Morphology, chemical structure and diffusion processes of
root surface of Er:YAG and Nd:YAG laser irradiation. J Clin Periodontol
2001;28:508-516.
Ge L H, Shu R, Shen M H. Effect of photodynamic therapy on IL-1beta and MMP-8
in gingival crevicular fluid of chronic periodontitis. Shanghai Kou Qiang Yi Xue.
2008;17:10-14.
Gold S I, Vilardi M A. Pulsed laser beam effects on gingiva. J Clin Periodontol.
1994;21:391-396.
Goldstein A, White J M, Pick R M. Clinical applications of the Nd:YAG laser. In:
Miserendido LJ, Pick RM (1995). Lasers in Dentistry, p.200.
Golub L M, Kleinberg I. Gingival crevicular fluid: a new diagnostic aid in
managing the periodontal patient. Oral Sci Rev. 1976;8:49-61
Haina D, Brunner R, Landthaler M, Braun-Falco O, Waidelich W. Animal
Experiments on Light-Induced Wound Healing. Biophysica, Berlin. 1973;35:227-230.
Harris D M, Yessik M. Therapeutic ratio quantifies laser antisepsis: ablation of
Porphyromonas gingivalis with dental lasers. Lasers Surg Med. 2004;35:206-213.
Hode L. The importance of the coherency. Photomed. Laser Surg. 2005;23:431-434.
Huang Y Y, Chen A C, Carroll J D, Hamblin M R. Biphasic dose response in low
level light therapy. Dose Response. 2010;7:358-383.
60. -
51
Hugoson A, Norderyd O. Has the prevalence of periodontitis changed during the last
30 Years? J Clin Periodontol. 2008;35:338-345.
Israel M, Cobb C M, Rossmann J A, Spencer P. The effects of CO2, Nd:YAG and
Er:YAG lasers with and without surface coolant on tooth root surfaces. An in vitro
study. J Clin Periodontol. 1997;24:595-602.
Iwase T, Saito T, Morioka T. Inhibitory effect of HeNe laser on dental plaque
deposition in hamsters. J Periodont Res. 1989;24:282-283.
Janoff A. Elastase in tissue injury. Ann Rev Med 1985;36:207-216.
Karu T I. Effects of visible radiation on cultured cells. Photochem Photobiol.
1990;52:1089-1098.
Karu T I. Ten Lectures of Basic Science of Laser Phototherapy. Prima Books AB,
Sweden. 2007. ISBN 978-91-976478-0-9.
Karu T I. Kalendo GS, Letokhov V S, Lobko VV. Biological action of low-intensity
visible light on HeLa cells as a function of the coherence, dose, wavelength, and
irradiation regime. Sov. J. Quantum Electron. 1982;12:1134-1138.
Karu T I, Kalendo G S, Letokhov V S, Lobko V V. Biological action of low-
intensity visible light on HeLa cells as a function of the coherence, dose, wavelength,
and irradiation regime. II. Sov. J. Quantum Electron. 1983;13:1169-1172.
Khullar S M, Brodin P, Barkvoll P, Haanaes H R. Preliminary study of low-level
laser for treatment of long-standing sensory aberrations in the inferior alveolar nerve. J
Oral and Maxillofac Surg. 1996;54:2-7.
Kiernicka M, Owczarek B, Galkowska E, Wysokinska-Miszczuk J. Comparison
of the effectiveness of the conservative treatment of the periodontal pockets with or
without use of laser biostimulation. Ann Univ Mariae Curie Sklodowska (Med)
2004;59:488-494.
61. 52
Kim I S, Cho T H, Kim K, Weber F E, Hwang S J. High power-pulsed Nd:YAG
laser as a new stimulus to induce BMP-2 expression in MC3T3-E1 osteoblasts. Lasers
Surg Med. 2010;42:510-518.
Kim Y D, Kim S S, Hwang D S, Kim S G, Kwon Y. H, Shin S H, Kim U K, Kim J
R, Chung I K. Effect of low-level laser treatment after installation of dental titanium
implant-immunohistochemical study of RANKL, RANK, OPG: an experimental study
in rats. Lasers Surg Med. 2007;39:441-450.
Kranendonk A, van der Reijden W, van Winkelhoff A, van der Weijden G. The
bactericidal effect of a Genius Nd:YAG laser. Int J Dent Hyg. 2010;8:63-67.
Kreisler M, Christoffers A B, Willershausen B, d'Hoedt B. Effect of low-level
GaAlAs laser irradiation on the proliferation rate of human periodontal ligament
fibroblasts: an in vitro study. J Clin Periodontol. 2003;30:353-358.
Lan W H, Liu H C. Treatment of Dentin Hypersensitivity by Nd:YAG Laser. J Clin
Laser Med Surg. 1996;14:89-92.
Liu C M, Hou L T, Wong M Y, Lan W H. Comparison of Nd:YAG laser versus
scaling and root planing in periodontal therapy. J Periodontol. 1999;70:1276-1282.
Lizarelli R F, Moriyama L T, Bagnato V S. Temperature response in the pulpal
chamber of primary human teeth exposed to Nd:YAG laser using a picosecond pulsed
regime. Photomed Laser Surg. 2006;24:610-615.
Loevschall H, Arenholt-Bindslev D. Effect of low level diode laser irradiation of
human oral mucosa fibroblasts in vitro. Lasers Surg Med. 1994;14:347-354.
Luomanen M. A comparative study of healing of laser and scalpel incision wounds in
rat oral mucosa. Scand J Dent Res. 1987;95:65-73.
62. -
53
Luza J, Hubacek J. In vitro He-Ne laser effect on some immunological functions of
the polymorphonuclears and monocytes in rabbits. Acta Univerity of Palacki Olomuc
Faculty of Medicine. 1996;140:43-46.
Löe H. The gingival index, the plaque index and the retention index system. J
Periodontol 1967;38:610-616.
McGuff E, Deterling R A Jr, Gottlieb L S. Tumoricidal effect of laser energy on
experimental and human malignant tumors. N Engl J Med. 1965;273:490-449.
Mester E, Szende B, Tota J. Die Wirkung der Laser-Strahlen auf den Haarwuchs der
Maus. Radiobiol. Radiother. 9: 621-626. Original paper: Mester E, Szende B, Tota JG.
Effect of laser on hair growth of mice. Kiserl Orvostud. 1967;19:628-631.
Miserendino L J, Levy G C, Abt E, Rizoiu I M. Histologic effects of a thermally
cooled Nd: YAG laser on the dental pulp and supporting structures of rabbit teeth. Oral
Surg Oral Med Oral Pathol Oral Radiol Endod. 1994;78:93-100.
Miyazaki A, Yamaguchi T, Nishikata J, Okuda K, Suda S, Orima K, Kobayashi
T, Yamazaki K, Yoshikawa E, Yoshie H. Effect of Nd:YAG and CO2 laser treatment
and ultrasonic scaling on periodontal pockets of chronic periodontitis patients. J
Periodontol 2003;74:175-180
Myers T D, McDaniel J D. The pulsed Nd:YAG dental laser: review of clinical
applications. J Calif Dent Assoc. 1991;19:25-30.
Nomura K, Yamaguchi M, Abiko Y. Inhibition of interleukin-1beta production and
gene expression in human gingival fibroblasts by low-energy laser irradiation. Lasers
Med Sci 2001;16:218-223.
Ozawa Y, Shimizu N, Abiko Y. Low-energy diode laser irradiation reduced
plasminogen activator activity in human periodontal ligament cells. Lasers Surg Med.
1997;21:456-463.
63. 54
Papapanou P N, Madianos P N, Dahlen G, Sandros J. "Checkerboard" versus
culture: a comparison between two methods for identification of subgingival
microbiota. European J Oral Sci. 1997;105:389-396.
Passarella S, Casamassima E, Molinari S, Pastore D, Quagliariello E, Catalano I
M, Cingolani A. Increase of proton electrochemical potential and ATP synthesis in rat
liver mitochondria irradiated in vitro by helium-neon laser.
FEBS Letters. 1984;175:95-99.
Pastore D, di Martino C, Bosco G, Passarella S. Stimulation of ATP synthesis via
oxidative phosphorylation in weak mitochondria irradiated with helium-neon laser.
Biochem Mol Biol Int. 1996;39:149-157.
Pejcic A, Zivkvic V. Histological examination of gingival treated with low-level laser
in periodontal therapy. J Oral Laser Appl. 2007;71:37-43.
Pejcic A, Kojovic D, Kesic L, Obradovic R. The effects of low level laser irradiation
on gingival inflammation. Photomed Laser Surg. 2010;28:69-74.
Pereira A N, Eduardo C de P, Matson E, Marques M M. Effect of low-power laser
irradiation on cell growth and procollagen synthesis of cultured fibroblasts. Lasers Surg
Med 2002;31:263-267.
Pires Oliveira D A, de Oliveira R F, Zangaro R A, Soares C P. Evaluation of low-
level laser therapy of osteoblastic cells. Photomed Laser Surg. 2008;26:401-404.
Pitaru S, McCulloch C A, Narayanan S A. Cellular origins and differentiation
control mechanisms during periodontal development and wound healing. J Periodontol
Res 1994;29:81-94.
64. -
55
Radvar M, Creanor S L, Gilmour W H, Payne A P, McGadey J. Foye R H,
Whitters C J, Kinane D F. An evaluation of the effects of an Nd:YAG laser on
subgingival calculus, dentine and cementum. An in vitro study. J Clin Periodontol.
1995;22:71-77.
Radvar M, MacFarlane T W, MacKenzie D, Whitters C J, Payne A P, Kinane D
F. An evaluation of the Nd:YAG laser in periodontal pocket therapy. Brit Dent J.
1996;80:57-62.
Ribeiro I W, Sbrana M C, Esper L A, Almeida A L. Evaluation of the effect of the
GaAlAs laser on subgingival scaling and root planing. Photomed Laser Surg.
2008;26:387-391.
Rochkind S, Nissan M, Lubart A. A single Transcutaneous Light Irradiation to
Injured Peripheral Nerve: Comparative Study with Five Different Wavelengths. Lasers
Med Sci. 1989;4:259-263.
Romanos G E, Henze M, Banihashemi S, Parsanejad H R, Winckler J, Nentwig G
H. Removal of epithelium in periodontal pockets following diode (980 nm) laser
application in the animal model: an in vitro study. Photomed Laser Surg.
2004;22:177-183.
Romeo U, Palaia G. Botti R, Leone V, Rocca J P, Polimeni A. Non-surgical
periodontal therapy assisted by potassium-titanyl-phosphate laser: a pilot study. Lasers
Med Sci 2010;7:738-746.
Rosner M, Caplan M, Cohen S, Duvdevani R, Solomon A, Assia E, Belkin M,
Schwartz M. Dose and temporal parameters in delaying injured optic nerve
degeneration by low-energy laser irradiation. Lasers Surg. Med. 1993;13:611-617.
Rydén H, Persson L, Preber H, Bergström J. Effect of low-energy laser on gingival
inflammation. Swed Dent J 1994;18:35-41.
65. 56
Safavi S M, Kazemi B, Esmaeili M, Fallah A, Modarresi A, Mir M. Effects of low-
level He-Ne laser irradiation on the gene expression of IL-1beta, TNF-alpha, IFN-
gamma, TGF-beta, bFGF, and PDGF in rat's gingiva. Lasers Med Sci.
2008;23:331-335.
Sawasaki I, Geraldo-Martins V R, Ribeiro M S, Marques M M. Effect of low-
intensity laser therapy on mast cell degranulation in human oral mucosa. Lasers Med
Sci. 2009;24:113-116.
Schwarz F, Aoki A, Becker J, Sculean A. Laser application in non-surgical
periodontal therapy: a systematic review. J Clin Periodontol. 2008;35:29-44.
Shimizu N, Yamaguchi M, Goseki T, Shibata Y, Takiguchi H, Iwasawa, Abiko Y.
Inhibition of prostaglandin E2 and interleukin 1-ß production by low-power laser
irradiation in stretched human periodontal ligament cells. J Dent Res.
1995;74:1382-1388.
Silness J, Löe H. Periodontal disease in pregnancy. II Correlation between oral
hygiene and periodontal conditions. Acta Odontol Scand 1964;22:121-131.
Silveira L B, Prates R A, Novelli M D, Marigo H A, Garrocho A A, Amorim J C,
Sousa G R, Pinotti M, Ribeiro M S. Investigation of mast cells in human gingiva
following low-intensity laser irradiation. Photomed Laser Surg. 2008;26:315-321.
Sjöström L, Friskopp J. Laser treatment as an adjunct to debridement of periodontal
pockets. Swed Dent J. 2002;26:51-57.
Sorsa T, Tjäderhane L, Salo T. Matrix metalloproteinases (MMPs) in oral diseases.
Oral Dis. 2004;10:311-318.
Spencer P, Cobb C M, McCollum M H, Wieliczka D M. The effects of CO2 laser
and Nd:YAG with and without water/air surface cooling on tooth root structure:
correlation between FTIR spectroscopy and histology. J Periodontal Res.
1996;31:453-462.
66. -
57
Tanaka H, Shimazu T, Sugimoto H, Yoshioka T, Sugimoto T. A sensitive and
specific assay for granulocyte elastase in inflammatory tissue fluid using L-
pyroglutamyl-L-propyl-L-valine-p-nitroanilide. Clinica Chimica Acta
1990:187;173-180.
Tervahartiala T, Pirilä E, Ceponis A, Maisi P, Salo T, Tuter G, Kallio P, Törnwall
J, Srinivas R, Konttinen Y T, Sorsa T. The in vivo expression of the collagenolytic
matrix metalloproteinases (MMP-2, -8, -13, and -14) and matrilysin (MMP-7) in adult
and localized juvenile periodontitis. J Dent Res. 2000;79:1969-1977.
Teughels W, Dekeyser C, van Essche M, Quirynen M. One-stage, full mouth
disinfection: Periodontol 2000;2009;39-51.
Tranaeus S, Shi X Q, Angmar-Månsson B. Caries risk assessment: methods
available to clinicians for caries detection. Community Dent Oral Epidemiol.
2005;33:265-273.
Wakao T, Yoshinaga E, Numabe Y, Kamoi K. Examination of periodontal disease
with gingival crevicular fluid. Correlation between capacitance and clinical findings.
Nippon Shishubyo Gakkai Kaishi. 1989;31:573-582.
White J M, Fagan M C, Goodis H E. Intrapulpal temperatures during pulsed Nd:
YAG laser treatment of dentin in vitro. J Periodontol 1994;65:255-259.
Xu M, Deng T, Mo F, Deng B, Lam W, Deng P, Zhang X, Liu S. Low-intensity
pulsed laser irradiation affects RANKL and OPG mRNA expression in rat calvarial
cells. Photomed Laser Surg. 2009;27:309-315.
Yilmaz S, Kuru B, Kuru L, Noyan U, Argun D, Kadir T. Effect of gallium arsenide
diode laser on human periodontal disease: a microbiological and clinical study. Lasers
Surg Med 2002;30:60-66.
Yu W, Naim J O, Lanzafame R J. The effect of laser irradiation on the release of
bFGF from 3T3 fibroblasts. Photochem Photobiol 1994;59:167-170.
67. 58
Yukna R A, Carr R L, Evans G H. Histologic evaluation of an Nd:YAG laser-
assisted new attachment procedure in humans. Int J Periodont Restor Dent.
2007;27:577-587.
Özcelik O, Cenk Haytac M, Kunin A, Seydaoglu G. Improved wound healing by
low-level laser irradiation after gingivectomy operations: a controlled clinical pilot
study. J Clin Periodontol. 2008a;35:250-254.
Özcelik O, Cenk Haytac M, Seydaoglu G. Enamel matrix derivative and low-level
laser therapy in the treatment of intra-bony defects: a randomized placebo-controlled
clinical trial. Clin Periodontol. 2008b;35:147-156.
